Therapeutic uses of smri peptides

ABSTRACT

The invention relates to the therapeutic use of a SMR1-peptide or a pharmaceutically active amount of said SMR1-peptide, for the preparation of a therapeutic composition for preventing or treating diseases wherein a modulation of the activity of a membrane metallopeptidase, notably a membrane-zinc metallopeptidase, is sought, in a mammal, specifically in a human.

[0001] In a first aspect, the invention relates to new therapeutic usesof a SMR1-peptide.

[0002] The inventors have previously characterized a new ratsubmandibular gland protein, named SMR1 (submandibular rat 1 protein),which has the structure of a prohormone and whose synthesis is underandrogen control (Rosinsky-Chupin et al., (1988), Proc. Natl. Acad. Sci.USA; 85(22):8553-7) and PCT Patent Application No. WO 90/03981). Thegene encoding SMR1 belongs to a new multigene family, the VCS family,which has been localized to rat chromosome 14, bands p21-p22 (Courty etal., (1996) Mol.Biol. Evol. 13(6):758-66; Rosinsky-Chupin et al., (1995)Mamm. Genome 6(2):153-4)) and for which some human gene members havebeen characterized (Isemura et al., (1997), J Biochem 121:1025-1030;Isemura et al. (1994) J Biochem 115:1101-1106; Isemura et al. (1979) JBiochem 86:79-86; Dickinson et al. (1996) Curr Eye Res 15:377-386). Thegene has an organization similar to a number of hormone precursor genes(Rosinsky-Chupin et al., (1990) DNA Cell. Biol. 9(8):553-9). SMR1 mRNAis expressed in a highly tissue-, age- and sex-specific manner in theacinar cells of the male rat submaxillary gland (SMG) and in theprostate (Rosinsky-Chupin et al., (1993) Histochem. Cytochem.41(11):1645-9).

[0003] It has been described that, in vivo, SMR1 is selectivelyprocessed at pairs of basic amino acid sites in a tissue- andsex-specific manner to give rise to mature peptide products, in a mannersimilar to the maturation pathway of peptide-hormone precursors (Rougeotet al., (1994) Eur. J Biochem. 219(3):765-73). The structurally relatedpeptides generated from SMR1 by cleavage at pairs of arginine residues(e.g. the undecapeptide: VRGPRRQHNPR; the hexapeptide: RQHNPR; and thepentapeptide: QHNPR) are in vivo selectively matured from the precursorafter processing at pairs of basic aminoacid residues by a paired basicaminoacid-converting enzyme, likely the Furine convertase,—differentially accumulated in a tissue-, age- and sex-related manner,and—locally as well as systemically released upon multifactorialneuroendocrine control (Rougeot et al, 1994).

[0004] In such a context, the final mature peptide generated fromSMR1,.named SMR1-Pentapeptide (SMR1-QHNPR), also named sialorphin, issynthesized predominantly in response to androgen steroids and isconstitutely released into the bloodstream in basal condition andacutely released in response to environmental stress, depending on thestate of activation of adrenoreceptors controlling the secretoryresponsiveness of the SMG.

[0005] In turn, the circulating SMR1-Pentapeptide is in vivo rapidly andselectively taken up by peripheral targets through specific bindingsites, predominantly within renal, bone and dental tissues.

[0006] The fact that the target sites of the peptide are mainlylocalized within the major tissues of ion capture, transport andregulation, gives evidence that SMR1-Pentapeptide might play a local andsystemic role in modulating mineral ion homeostasic process, in vivo.Furthermore, associated with the fact that the androgen-regulatedSMR1-Pentapeptide is upon environmental stress acutely secreted, thesefindings led the inventors to postulate that this SMG-specific signalingpeptide might participate in mediating integrative reestablishment ofdynamic homeostatic responses: to stressful situations within malerat-specific behavioral characteristics such as aggressive and/or sexualintercourses, and in relation to female-specific physiologicalcharacteristics such as pregnancy and lactation.

[0007] WO 98/37 100 discloses that the maturation products of the SMR1protein, specifically the peptide of structural formula XQHNPR,recognize specific target sites in organs that are deeply involved inthe mineral ion concentration. This discovery has led the inventors toassign to the SMR1-peptide (especially the SMR1-pentapeptide,hexapeptide or undecapeptide) an active role in the regulation of themetal ion concentrations in the body fluids and tissues, and thus atherapeutic role of these peptides in all the metabolic disordersrelated to a mineral ion imbalance.

[0008] Namely, the therapeutic peptides disclosed therein are useful fortreating or preventing bone, teeth, kidney, intestine, pancreas,stomach, or salivary gland disorders caused by a mineral ion imbalancein the body fluids or tissues, namely hyper- or hypo-parathyroidism,osteoporosis, pancreatitis, submandibular gland lithiasis,nephrolithiasis or osteodystrophy.

[0009] On the basis of the hypothesis mentioned above, a behavioralpharmacological approach has been undertaken. SMR1-peptide, especiallySMR1-Pentapeptide was found to induce a dose-dependent improvement onthe sexual behavior of adult male rats with a loss of the aggressiveimpulse behavior seen in control rats PCT patent application WO 01/00221).

[0010] To elucidate the pathways that have taken place in theSMR1-peptide action, one of the essential steps was to investigate themolecular characteristics of the peptide-receptor sites. The isolationof the membrane binding site accessible to the systemic administrationor radiolabelled SMR1-Pentapeptide, especially within the renal outermedulla has been achieved. The identification of its amino-acid sequencehas revealed that the cell surface molecule which binds the peptide invivo, is a membrane metallopeptidase and more specifically a mammaliantype II integral membrane zinc-containing endopeptidase, i.e. NeutralEndoPeptidase 24-11 or NEP, also named Enkephalinase that belongs to theNeprilysin subfamily, which plays critical role in the functionalpotency of various peptidergic signals. Moreover, the in vivo directinteraction of rat kidney NEP and SMR1-Pentapeptide was demonstrated invitro using purified rabbit kidney NEP.

[0011] Furthermore, at the level of whole rat body a good (topologicaland kinetical) correspondence was found in vivo between the distributionof target organs accessible to circulating radiolabelled SMR1-Pentapeptide and that of known synthetic NEP inhibitor (3HHACBO-Gly)(Sales et al, (1991) Regulatory Peptides 33, 209-22). Otherwise, anumber of observations argues for the hypothesis that SMR1-peptide is aSMG-derived natural modulator, especially an inhibitor, of the NEPactivity:

[0012] 1—the SMR1 -Pentapeptide tissue uptake was found to bepharmacokinetically and biochemically stable in vivo,

[0013] 2—the SMR1-peptide does not share the residues required to be aNEP substrat, seeing that the NEP preferentially cleaves peptidesbetween the X-Phe bond, and

[0014] 3—the SMR1-Pentapeptide has strong zinc-chelating group, whichhas been designed for the potent synthetic NEP inhibitors.

[0015] In view of the numerous physiological NEP substrates (namely thepeptide hormones: Enkephalins, Substance P, Bradykinin, Angiotensin IIand atrial natriuretic peptide), physiological consequences of theNEP/SRM1-peptide interaction are expected to impact on the control ofcentral and peripheral pain perception, inflammatory phenomena, arterialtone and/or mineral exchange (Roques et al, 1993 infra).

[0016] Neutral endopeptidase, NEP 24-11, is distributed both in nervousand peripheral tissues of mammals, and in the periphery it isparticularly abundant in the kidney and placenta. In these tissues thecell-surface metallopeptidase NEP participates in the postsecretoryprocessing and metabolism of neuropeptides, systemic immunoregulatorypeptides and peptide-hormones. By controlling the active levels ofcirculating or secreted regulatory peptides, NEP modulates theirphysiological receptor-mediated action. Hence, the membrane-anchored NEPis involved in regulating the activity of: potent vasoactive peptidessuch as Substance P, Bradykinin (BK), Atrial Natriuretic peptide (ANP),and Angiotensin II (AII); potent inflammatory/immunoregulatory peptidessuch as Substance P and BK and fMet-Leu-Phe (fMLP); potent opioidneuropeptides such as Met and Leu-Enkephalins CEnk) and potent mineralexchange and fluid homeostasis regulatory peptides such as ANP, C-typeNatriuretic Peptide (CNP) and B-type Natriuretic Peptide (BNP). Howeverthe levels of these peptides are changed through the NEP-inducedformation/degradation only in regions where they are tonically releasedor where their release is triggered by a stimulus.

[0017] From an integrative point of view, the NEP biological activity isto control the active levels of peptidergic signals involved in arterialtension regulation, in inflammatory phenomena and in water-mineralhomeostasis, as well as, in the control of pain processing. From aclinical point of view, this substantiates the fact that NEP is animportant drug target in various disease states. For example, byinhibiting NEP, thereby increasing the levels and duration of action ofcentral or peripheral endogenous opioids, an analgesic or antidiarrhealagent could be obtained, or by inhibiting endogenous AII formation andsubstance P, BK and ANP inactivation, antihypertensive, natriuretic anddiuretic agents could be obtained. The main advantage of modifying theconcentrations of endogenous peptides by use of NEP inhibitors is thatthe pharmacological effects are induced only at receptor stimulated bythe natural effectors, and are critically dependent on the tonic orstimulus-evoked release of the natural effectors happening uponenvironmental, behavioral and physiopathological stressful situations(Roques et al, (1993) Pharmacological Reviews 45, 87-146). It isimportant to stress that in such stressful context, the naturalpotential NEP-modulator, SMR1 -peptide, will be also acutely andtonically released, distributed and taken up by its systemic targettissues, especially by the renal NEP sites Rougeot et al, 1997).Thereby, the SMR1 -peptide would be in vivo kinetically bioavailable tomodulate NEP activity and so to optimize the local and systemicinflammatory, pressor and/or ion homeostatic responses to stress. Theintegrative point of view is in concordance with the assumption thatcirculating Submaxillary Gland (SMG)-derived factors might participatein integrative reestablishment of homeostatic responses to physiologicalor pathological “stress states” (injury, trauma or infection), ratherthan contribute to the resting homeostatic steady state (Rougeot et al,(2000) Peptides 21, 443-55).

[0018] From a general point of view, evidence of a physiologicalsignificance demonstrates the existence of a Cervical Sympathetic Trunk(CST)-SMG neuroendocrine axis that plays an integral role inphysiological adaptations and contributes to the maintenance ofhomeostasis in mammals, especially under the “stress conditions” seen inrodents with tissue damage, inflammation, and aggressive behavior. Thedata gathered in the laboratory provide convincing evidence thatSMR1-peptide is a novel signaling mediator, adapted to the sex, andspecies-specific environmental, behavioral and physiologicalcharacteristics, tonically and dynamically mobilized upon urgentsituations in the way to optimize both local and systemic nociceptive,inflammatory, pressor and/or ion homeostatic responses, throughregulation of the membrane-bound NEP activity. Otherwise, theSMR1-peptide, which is to date the first natural regulator of theperipheral NEP activity identified, seems to be designed as a new classof therapeutic molecules as this metallopeptidase is well-conservedespecially between rat, rabbit and human species with sequence homology≧90%.

[0019] The evidence provided by the inventors together with the strikinghomology with the NEP sequences between species further suggest that theSMR1-peptide may act as natural modulator/inhibitor of membranemetallopeptidases, notably zinc metallopeptidases (GenBank Access numberP 08473, Malfroy et al, (1988) FEBS Lett. 229(1), 206-210; NP 258428,Bonvouloir et al, (2001) DNA Cell Biol. 20(8), 493-498; NP 036740,Malfroy et al. (1987) Biochem Biophys Res Commun 144, 59-66).

[0020] Examples of mammalian membrane metallopeptidases besides NEP areECE (Endothelin-Converting Enzymes), in particular ECE1 and ECE2, theerythrocyte cell-surface antigen KELL and the product of PEX geneassociated with X-linked hypophosphatemic rickets, as well as ACE(Angiotensin Converting Enzyme) and APN (Aminopeptidase N).

[0021] Inhibition of ACE and/or ECE has a significant application in thetreatment of hypertension and the prevention and treatment ofatherosclerosis.

[0022] Inhibition of APN in conjunction with NEP has significantapplication in the treatment of pain.

[0023] Inhibition of related membrane metallopeptidases has therapeuticeffects in the treatment of tumors, namely ovarian, colorectal, brain,lung, pancreas, gastric and melanoma cancers, and reducing the incidenceof metastasis, atherosclerosis and/or hypertension. inhibitions ofrelated membrane metallopeptidases has also therapeutic effects in paincontrolling. Such antinociceptive effects on acute pain are analgesiceffects but also effects on chronic inflammatory pain such as arthritisor inflammatory bowel disease.

[0024] Furthermore, inhibition of bacterial or viral metallopeptidase isexpected to have anti-infection effects.

[0025] Metallopeptidases playing an important role in pathogen hosttissue invasion and immunological and inflammatory processes, forexample those of Streptococcus pyogenes, Pseudomonas aeruginosa,Porphyromonas gingivalis and Legionella pneumophila.

[0026] Furthermore, bacterial metallopeptidases, especiallyzinc-metallopeptidases play an important role in the diseases caused byproteolytic toxins, such as the toxins of B. anthracis (Anthrax Lethalfactor) and the neurotoxins of C. tetanum and botulinum.

[0027] Other metallopeptidases play an important role in variousinfections such as infections caused by HIV (FR 2 707 169).

[0028] The importance of proteinase inhibitors for the treatment ofbacterial or viral diseases may be found in J. Potempa, J. Travis,(Proteinases as virulence factors in bacterial diseases and as potentialtargets for therapeutic interaction with proteinase inhibitors. Inproteases as targets for therapy. 99, 159-188—Eds K. Helm, B. D. Korantand J. C. Cheronis—Spinger Handbook Exp. Pharm. 140).

[0029] The different roles of metallopeptidases are disclosed in Turneret al, (2001) Bioessays, 23, 261-9; Kenny et al, (1977) Proteinases inmammalian cells and tissues); Kenny et al, (1987) Mammalian ectoenzymes;Beaumont et al, (1996) zinc metallopeptidases in health and disease,105-129).

[0030] A first subject-matter of the invention is thus the therapeuticuse of a SMR1-peptide or a pharmaceutically active amount of saidSMR1-peptide, for the preparation of a therapeutic composition forpreventing or treating diseases wherein a modulation of the activity ofa membrane metallopeptidase, notably a membrane zinc metallopeptidase,is sought, in a mammal, specifically in a human.

[0031] Another object matter of the invention is the therapeutic use ofan agent such as a biologically active derivative of SMR1-peptide formodulating the interaction between the endogenous SMR1-peptide and saidmembrane metallopeptidase. Said modulation is a kinetical and/ormolecular one.

[0032] “Endogenous” refers to a molecule (herein a SMR1-peptide) that isnaturally expressed or matured in tissues of a patient to be treated.

[0033] The invention further relates to the use of an agent thatmodulates the interaction between endogenous SMR1 protein or peptide anda membrane metallopeptidase for the preparation of a therapeuticcomposition for preventing or treating diseases wherein a modulation ofthe activity of said membrane metallopeptidase is sought.

[0034] The present invention concerns more specifically the therapeuticuse of the SMR1-peptide or a pharmaceutically active amount of aSMR1-peptide, for the preparation of a medicament for preventing ortreating diseases wherein modulation of NEP-induced degradation ofNEP-sensitive peptides is sought, in a mammal, specifically in human.

[0035] As used in the present specification, SMR1-peptide means the SMR1protein, a peptide generated from SMR1, also called a maturation productof the SMR1 protein, or one of the biologically active derivatives ofsaid protein or said maturation product.

[0036] In a preferred embodiment, the SMR1-peptide is a compound ofstructural formula (1):

X₁QHX₂X₃X₄

[0037] wherein X₁ denotes a hydrogen atom or X₁ represents an amino acidchain selected from the following: X₁═R or G, X₁═RR, or X₁═PRR, orX₁═GPRR, or X₁═RGPRR, or X₁═VRGPRR, X₂ denotes N, G or D, X₃ denotes Por L and X₄ denotes R or T.

[0038] Preferred peptides comprise peptides of sequence:

[0039] QHNPR, RQHNPR and VRGPRRQHNPR from Ratus norvegius,

[0040] QHNLR and RQHNLR from Ratus ratus,

[0041] GQHGPR and GQHDPT from mouse.

[0042] In the above aminoacid sequences:

[0043] Q represents Glutamine,

[0044] H represents Histidine,

[0045] N represents Asparagine,

[0046] G represents Glycine,

[0047] P represents Proline,

[0048] R represents Arginine,

[0049] L represents Leucine,

[0050] T represents Threonine,

[0051] D represents Aspartic acid, and

[0052] V represents valine.

[0053] “Biologically active derivatives” of the SMR1-peptide refer tofunction-conservative variants, homologous proteins and peptidomimetics,as well as a hormone, an antibody or a synthetic compound, (i.e. eithera peptidic or non peptidic molecule) that preferably retain the bindingspecificity and/or physiological activity of the parent peptide, asdefined below. They preferably show an ability to bind a membranemetallopeptidase, more particularly NEP. Such binding activity may bereadily determined by binding assays, e.g. by labeling the SMRLderivative or by competition assay with a conventional NEP substrate,optionally labeled.

[0054] “Function-conservative variants” are those in which a given aminoacid residue in a protein has been changed without altering the overallconformation and function of the polypeptide, including, but not limitedto, replacement of an amino acid with one having similar properties(such as, for example, polarity, hydrogen bonding potential, acidic,basic, hydrophobic, aromatic, and the like). Amino acids with similarproperties are well known in the art. For example, arginine and lysineare hydrophilic-basic amino acids and may be interchangeable. Similarly,isoleucine, a hydrophobic amino acid, may be replaced with leucine orvaline. Such changes are expected to have little or no effect on theapparent molecular weight or isoelectric point of the protein orpolypeptide. Amino acids other than those indicated as conserved maydiffer in a protein or enzyme so that the percent protein or amino acidsequence similarity between any two proteins of similar function mayvary and may be, for example, from 70% to 99% as determined according toan alignment scheme such as by the Cluster Method, wherein similarity isbased on the MEGALIGN algorithm. A “function-conservative variant” alsoincludes a polypeptide or enzyme which has at least 60% amino acididentity as determined by BLAST or FASTA algorithms, preferably at least75%, most preferably at least 85%, and even more preferably at least90%, and which has the same or substantially similar properties orfunctions as the native or parent protein or enzyme to which it iscompared.

[0055] “Allelic variants” are more particularly encompassed, asdescribed in greater details below.

[0056] As used herein, the term “homologous” in all its grammaticalforms and spelling variations refers to the relationship betweenproteins that possess a “common evolutionary origin,” including proteinsfrom superfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al, Cell 50:667, 1987). Such proteins have sequence homology, asreflected by their sequence similarity, whether in terms of percentsimilarity or the presence of specific residues or motifs at conservedpositions.

[0057] Accordingly, the term “sequence similarity” in all itsgrammatical forms refers to the degree of identity or correspondencebetween amino acid sequences of proteins that may or may not share acommon evolutionary origin (see Reeck et al., supra). However, in commonusage and in the instant application, the term “homologous,” whenmodified with an adverb such as “highly,” may refer to sequencesimilarity and may or may not relate to a common evolutionary origin.

[0058] In a particular embodiment, two amino acid sequences are“substantially homologous” or “substantially similar” when greater than80% of the amino acids are identical, or greater than about 90% aresimilar and functionally identical. Preferably, the similar orhomologous sequences are identified by alignment using, for example, theGCG (Genetics Computer Group, Program Manual for the GCG Package,Version 7, Madison, Wis.) pileup program, or any of the programsdescribed above (BLAST, FASTA, etc.).

[0059] Natural NEP substrates are mainly the peptide hormones:Enkephalins, Substance P, Bradykinin, Angiotensin II and AtrialNatriuretic Peptide which play key role in the control of central andperipheral pain perception, inflammatory phenomena, mineral exchangeand/or arterial tone.

[0060] More particularly, one object of the present invention is the useof the above described therapeutic peptides as analgesic agents byinhibiting NEP at peripheral, spinal and/or supraspinal levels andthereby increasing the levels and duration of action of central orperipheral endogenous opioids, including enkephalins.

[0061] Another object is the use of the above described peptides asantidiarrheal agents.

[0062] Another object is the use of the above described peptides asantihypertensive, natriuretic and diuretic agents by inhibitingendogenous AII formation and substance P, BK and ANP inactivation.

[0063] A further object is the use of the above described peptides as anagent for preventing or treating atherosclerosis.

[0064] Another object is the use of the above described peptides as anagent for the treatment of pain including chronic inflammatory pain,such as arthritis or inflammatory bowel disease.

[0065] Another object is the use of the above described peptides as anagent for controlling immuno-inflammatory responses.

[0066] Another object is the use of the above described peptides as anagent for preventing or treating the processes of malignant cellproliferation and dissemination.

[0067] Another object of the present invention is the use of the abovedescribed peptides as a substitute in the treatment of drug abuse,notably morphine drug abuse.

[0068] Indeed, studies have suggested that the vulnerability to drugabuse and the development of reward and drug dependence is at least inpart, a result of pre-existent or induced modifications and/or defect ofthe endogenous opioid system. In this regard, using SMR1-peptide topotentiate the effects of endogenous enkephalins will reduce the variousside-effects (somatic signs of withdrawal) produced by interruption ofchronic morphine or heroin administration.

[0069] Still another object of the invention is the use of the abovedescribed peptides for treating infections such as bacterial or viraldiseases.

[0070] For purposes of the invention, the term “mammal” is used in itsusual taxonomic sense and specifically includes humans.

[0071] For purposes of the invention, a “peptide” is a moleculecomprised of a linear array of amino acid residues connected to eachother in the linear array by peptide bonds. Such linear array mayoptionally be cyclic, i.e., the ends of the linear peptide or the sidechains of amino acids within the peptide may be joined, e.g., by achemical bond. Such peptides according to the invention may include fromabout three to about 500 amino acids, and may furher include secondary,tertiary or quaternary structures, as well as intermolecularassociations with other peptides or other non-peptide molecules. Suchintermolecular associations may be through, without limitation, covalentbonding (e.g., through disulfide linkages), or through chelation,electrostatic interactions, hydrophobic interactions, hydrogen bonding,ion-dipole interactions, dipole-dipole interactions, or any combinationof the above.

[0072] Preferred peptides according to the invention comprise an aminoacid sequence selected from the group consisting of:

[0073] Glp-His-Asn-Pro-Arg [SEQ ID NO. 1]

[0074] Gln-His-Asn-Pro-Arg [SEQ ID NO. 2]

[0075] Arg-Gln-His-Asn-Pro-Arg [SEQ ID NO. 3]

[0076] Val-Arg-Gly-Pro-Arg-Arg-Gln-His-Asn-Pro-Arg [SEQ ID NO 4]

[0077] Gln-His-Asn-Leu-Arg [SEQ ID NO 5]

[0078] Arg-Gln-His-Asn-Leu-Arg [SEQ ID NO 6]

[0079] Gly-Gln-His-Gly-Pro-Arg [SEQ ID NO 7]

[0080] Gly-Gln-His-Asp-Pro-Thr [SEQ ID NO 8]

[0081] wherein the sequences are shown in N to C configuration, andwherein Glp is pyroglutamate, Gln is glutamine, His is histidine, Asn isasparagine, Pro is proline, Arg is Arginine, Gly is Glycine, Val isValine, Leu is Leucine, and Thr is Threonine.

[0082] In these peptides, by N-terminal cyclization/decyclization, Glpand Gln interconvert.

[0083] In addition, certain preferred peptides according to theinvention comprise, consist essentially of, or consist of an allelicvariant of a peptide shown in any of SEQ ID NO. 1-8. As used herein, an“allelic variant” is a peptide having from one to two amino acidsubstitutions from a parent peptide, but retaining the bindingspecificity and/or physiological activity of the parent peptide. As usedherein, “retaining the binding specificity of the parent peptide” meansbeing able to bind to a monoclonal or polyclonal antibody that binds toone of the peptides shown in SEQ ID NOS. 1-8 with an affinity that is atleast one-tenth, more preferably at least one-half, and most preferablyat least as great as that of one of the actual peptides shown in SEQ IDNOS. 1-8. Determination of such affinity is preferably conducted understandard competitive binding immunoassay conditions (Rougeot et al., (E.J. B. 219(3) 765-773). “Retaining the physiological activity of theparent peptide” means retaining the ability of any one of the peptidesshown in SEQ ID NOS. 1-8 to bind and to modulate NEP-activity and so tooptimize the local and systemic nociceptive, inflammatory, pressor,and/or ion homeostatic responses to stress. Determining whether suchactivity is modulated is futther described later in this specification.The term “allelic variants” is specifically intended to include anyhuman functional homologs of the peptides set forth in SEQ ID NOS. 1-8which do not have the identical amino acid sequence thereof.

[0084] Peptides according to the invention can be convenientlysynthesized using art recognized techniques (see e.g., Merrifield, J.Am. Chem. Soc. 85: 2149-2154).

[0085] Also part of the invention are preferred peptidomirneticsretaining the binding specificity and/or physiological activity of theparent peptide, as described above. As used herein, a “peptidoniimetic”is an organic molecule that mimics some properties of peptides,preferably their binding specificity and/or physiological activity.Preferred peptidomimetics are obtained by structural modification ofpeptides according to the invention, preferably using unnatural aminoacids, D amninoacid instead of L aminoacid, conformational restraints,isosteric replacement, cyclization, or other modifications. Otherpreferred modifications include without limitation, those in which oneor more amide bond is replaced by a non-amide bond, and/or one or moreamino acid side chain is replaced by a different chemical moiety, or oneof more of the N-terminus, the C-terminus or one or more side chain isprotected by a protecting group, and/or double bonds and/or cyclizationand/or stereospecificity is introduced into the amino acid chain toincrease rigidity and/or binding affinity.

[0086] Still other preferred modifications include those intented toenhance resistance to enzymatic degradation, improvement in thebioavailability in particular by nervous, intestinal, placental andgonad tissues and more generally in the pharmacokinetic properties andespecially comprise:

[0087] protecting the NH₂ and COOH hydrophilic groups by esterification(COOH) with lipophilic alcohols or by amidation (COOH) and/or byacetylation (NH₂) or added carboxyalkyl or aromatic hydrophobic chain atthe NH₂ terminus;

[0088] retroinversion or reduction isomers of the CO—NH amide bonds ormethylation (or ketomethylene, methyleneoxy, hydroxyethylene) of theamide functions;

[0089] substitution of L aminoacids for D aminoacids;

[0090] dimerisation of amino acid peptide chain.

[0091] All of these variations are well known in the art. Thus, giventhe peptide sequences disclosed herein, those skilled in the art areenabled to design and produce peptidomimetics having bindingcharacteristics similar to or superior to such peptides (see e.g.,Horwell et al., Bioorg. Med. Chem. 4: 1573 (1996); Liskamp et al., Recl.Trav. Chim. Pays- Bas 1: 113 (1994); Gante et al., Angew. Chem. Int. Ed.Engl. 33: 1699 (1994); Seebach et al., Helv. Chim. Acta 79: 913 (1996)).

[0092] The peptides used according to the present invention may beprepared in a conventional manner by peptide synthesis in liquid orsolid phase by successive couplings of the different amino acid residuesto be incorporated (from the N-terminal end to the C-terminal end inliquid phase, or from the C-terminal end to the N-terminal end in solidphase) wherein the N-terminal ends and the reactive side chains arepreviously blocked by conventional groups.

[0093] For solid phase synthesis the technique described by Merrifieldmay be used in particular. Alternatively, the technique described byHoubenweyl in 1974 may also be used.

[0094] For more details, reference may be made to WO 98/37 100.

[0095] The peptides used in the therapeutic method according to thepresent invention may also be obtained using genetic engineeringmethods. The nucleic acid sequence of the cDNA encoding the complete 146amino acid SMR1 protein has been described in the PCT Patent ApplicationNo. WO 90/03891 (Rougeon et al.) For the biologically active peptidederivatives of the SMR1-peptide, for example a derivative of X₁QHX₂X₃X₄,a person skilled in the art will refer to the general literature todetermine which appropriate codons may be used to synthetize the desiredpeptide.

[0096] The methods that allow a person skilled in the art to select andpurify the biologically active derivatives that bind to the same targetsand have an agonist or an antagonist biological activity of theSMR1-peptide of the invention are described hereunder.

[0097] The biologically active derivative of the SMR1-peptide may be aprotein, a peptide, a hormone, an antibody or a synthetic compound whichis either a peptide or a non peptidic molecule, such as any compoundthat can be synthesized by the conventional methods of organicchemistry.

[0098] Selection of the biologically active derivatives of theSMR1-peptide of the invention is performed both in assessing the bindingof a candidate ligand molecule to the NEP binding site for the QHNPRpentapeptide, and in determining the metabolic changes induced by thiscandidate molecule on its target, such as the synthesis and/or releaseof the primary or secondary messenger metabolites as a result of atransduction signal via the protein kinases or adenylate cyclase and theactivation of a protein of the G family or the variation of theenzymatic activity of NEP, specifically on the metabolism of natural NEPsubstrates.

[0099] Binding assays of the candidate molecule are generally performedat 4° C. to 25° C. or 37° C. In order to facilitate the reading of thehereinafter described protocol, QHNPR pentapeptide is also used insteadof or in competition with a biologically active derivative candidatemolecule.

[0100] Accordingly, another object of the present invention is a processfor screening ligand molecules that specifically bind to the NEP bindingsite for the QHNPR pentapeptide, comprising the steps of:

[0101] a) preparing a cell culture or preparing an organ specimen or atissue sample (cryosections or slices or membrane preparations or crudehomogenates) containing NEP binding sites for the QIMR pentapeptide;

[0102] b) adding the candidate molecule to be tested in competition withhalf-saturating concentration of labeled pentapeptide;

[0103] c) incubating the cell culture, organ specimen or tissue sampleof step a) in the presence of the candidate molecule during a timesufficient and under conditions for the specific binding to take place;

[0104] d) quantifying the label specifically bound to the cell culture,organ specimen or tissue sample in the presence of variousconcentrations of candidate molecule (preferably 10⁻¹⁰ to 10⁻⁵ M).

[0105] In said above process, a half-saturating concentration is theconcentration of the labelled QHNPR pentapeptide which binds 50% of theNEP binding sites.

[0106] This process also allows to define the relative affinity of thecandidate molecule compared to the QHNPR affinity.

[0107] Another object of the present invention is a process fordetermining the relative affinity of ligand molecules that specificallybind to the NEP binding sites for the QHNPR pentapeptide comprising thesteps a), b), c) and d) of the above process for each candidate moleculeand further comprising the step e) of comparing the affinity of eachcandidate molecule quantified in step d) to the one of the othercandidate molecules.

[0108] Another object of the present invention is a process fordetermining the affinity of ligand molecules that specifically bind tothe NEP binding site for the QHNPR pentapeptide, comprising the stepsof:

[0109] a) preparing a cell culture or preparing an organ specimen or atissue sample (cryosections or slices or membrane preparations or crudehomogenates) containing NEP binding sites for the QHNPR pentapeptide;

[0110] b) adding the candidate molecule which has previously beenlabeled with a radioactive or a nonradioactive label;

[0111] c) incubating the cell culture, organ specimen or tissue sampleof step a) in the presence of the labeled candidate molecule during atime sufficient and under conditions for the specific binding to takeplace; and

[0112] d) quantifying the label specifically bound to the cell culture,organ specimen or tissue sample in the presence of variousconcentrations of the labeled candidate molecule (preferably 10⁻¹⁰ to10⁻⁵M).

[0113] The candidate ligand molecule may be radioactively labeled (³²P,³⁵S, ³H, ¹²⁵I etc.) or nonradioactively labeled (biotin, digoxigenin,fluorescein etc.)

[0114] Thus, the present invention also pertains to a process forscreening ligand molecules that possess an agonist biological activityon the NEP binding site of the QHNPR pentapeptide, comprising the stepsof:

[0115] a) preparing a cell culture or preparing an organ specimen or atissue sample (cryosections or slices or membrane preparations or crudehomogenates) containing NEP binding sites for the QHNPR pentapeptide;

[0116] b) incubating the cell culture, organ specimen or tissue sampleof step a) at concentrations allowing measurement of NEP enzymaticactivity under initial velocity conditions as defined by the method ofExample 1 (Material and methods) in the presence of the candidatemolecule (preferably 10⁻¹⁰-10⁻⁵ M), a half-saturating concentration ofQHNPR and a NEP substrate during a time sufficient for the hydrolysis ofthe NEP substrate to take place under initial velocity conditions;

[0117] c) quantifyig the activity of the NEP present in the biologicalmaterial of step a) by measuring the levels of NEP substrate hydrolysis,respectively in the presence or in the absence of the candidate ligandmolecule and in the presence or in the absence of QHNPR.

[0118] In said above process, a half-saturating concentration is theconcentration of the QHNPR pentapeptide which reduces by half thedegradation of the NEP substrate.

[0119] Another object of the present invention comprises a process forscreening ligand molecules that possess an antagonist biologicalactivity on the NEP binding site of the QHNPR pentapeptide, comprisingthe steps of:

[0120] a) preparing a cell culture or preparing an organ specimen or atissue sample (cryosections or slices or membrane preparations or crudehomogenates) containing NEP binding sites for the QBNPR pentapeptide;

[0121] b) incubating the cell culture, organ specinen or tissue sampleof step a) at concentration allowing measurement of NEP enzymaticactivity under initial velocity conditions in the presence of asubmaximal concentration of the XQHNPR peptide, specifically the QHNPRpeptide and a NEP substrate, in the presence of the candidate moleculeduring a time sufficient for the hydrolysis of the NEP substrate to takeplace under initial velocity conditions;

[0122] c) quantifying the activity of the NEP present in the biologicalmaterial of step a) by measuring the levels of NEP substrate hydrolysis,respectively in the presence or in the absence of the candidate ligandmolecule and in the presence or in the absence of QHNPR.

[0123] In a preferred embodiment of said above process, a submaximalconcentration is a concentration of pentapeptide which reduces by atleast 50% and preferably by at least 75% the degradation of thesubstrate.

[0124] As mentioned above, another metabolic assay in order to assessthe agonist or the antagonist activity of the candidate ligand moleculecomprises the incubation of the ligand candidate in the presence of aprimary cell culture or established cell line or tissue sample of rat,mouse or human origins and an endogenous or exogenous NEP substrate anddetermining, either or both quantitatively and qualitatively, thehydrolysis of the NEP substrate.

[0125] A preferred tissue sample that is used in the screening methodsaccording to the present invention is a membrane preparation or slicesof spinal cord from rats, a tissue known to be appropriated for NEPactivity measurement.

[0126] Other preferred tissue samples that can be used in the screeningmethods according to the present invention are all peripheral tissuepreparations that are known to be enriched in NEP-peptidase and/or to betargets for SMR1-peptide, for example rat renal outer medulla, placenta,testis, prostate and bone and dental tissues. In addition, such aprocedure can also be applied to tissues and/or cells of mammals (e.g.mouse) and especially human origin or cell lines transfected with humanNEP cDNA, for example MDCK, HEK or COS cells first transfected withhuman NEP cDNA.

[0127] Preferred biologically active derivatives of SMR1-peptide andspecially of X₁QHX₂X₃X₄ of the therapeutic composition according to thepresent invention have better pharmacodynamic properties than theendogenous natural or synthetic X₁QHX₂X₃X₄ peptide, and thus possess alonger in vivo half-life as compared to their natural counterparts and abetter bioavailability in a given tissue/space, especially in nervous,intestine, placental and gonad tissues.

[0128] The above-described biologically active derivatives, are also anobject of the present invention.

[0129] Thus, the invention also relates to the SMR1 maturation productsand the biologically active derivatives of the SMR1 protein or of itsmaturation products that can be selected according to the screeningprocesses hereinbefore described, provided that they have not thestructure of formula (1) above. Indeed, also excluded, is the 146 aminoacid protein constituting the SMR1 protein itself (PCT PatentApplication N^(o) WO 90/03981). However, the therapeutic use of thesemolecules that are excluded as such of the present invention, is a mainobject of the instant invention.

[0130] Another object of the present invention is a biologically activederivative of the SMR1-peptide characterized by its capacity either toincrease or decrease a metallopeptidase activity or to prevent thenormal interaction between the SMR1-peptide and said metallopeptidase.Preferably, said metallopeptidase is a membrane-zinc metallopeptidase.More preferably, said membrane-zinc metallopeptidase is NEP.

[0131] The biologically active derivatives of SMR1-peptide socharacterized also include SMR1 protein maturation products, providedthat they do not have the structure of formula (1) above.

[0132] The SMR1 protein or its maturation products and the biologicallyactive derivatives of the SMR1 protein or of its maturation productsused in the therapeutic compositions according to the present inventionhave been, in a preferred embodiment, selected firstly according totheir ability to bind to the same targets as the X₁QHX₂X₃X₄,specifically QHNPR peptide, and secondly by their capacity to modulatehydrolysis of substrate of a metallopeptidase for example the NEP invitro or in vivo.

[0133] By “modulate”, it is understood that said SMR1-peptide has thecapacity either to increase or decrease (inhibit) the metallopeptidaseactivity or to prevent the normal interaction between the SMR1-peptideand the said metallopeptidase.

[0134] The present invention also deals with the use of therapeuticcompositions comprising an effective amount of the SMR1-peptide.

[0135] In the methods according to the invention, the peptides orpeptidomimetics according to the invention may be administered by any ofa variety of means. In certain preferred embodiments, administration maybe parenteral, most preferably intravenous. In other preferredembodiments, administration may be intranasal, oral, sublingual,transmucosal, intrarespiratory, or through an inert or iontophoreticpatch.

[0136] Dosages of the peptide or peptidomimetic to be administered willdepend on the particular patient, the condition, and the route ofadministration, and can be determined empirically by the reduction orelimination linked to the pathological disorders listed above inresponse to an elevating dosage regimen. Preferred dosages are fromabout 0.1 μg/kg to about 1 mg/kg, more preferably from about 1 μg/kg toabout 100 μg/kg, and most preferably from about 1 μg/kg to about 50μg/kg.

[0137] In certain preferred embodiments, the peptide or peptidomimeticaccording to the invention is administered together with a secondpharmaceutical, wherein the second pharmaceutical agent is present in anamount insufficient to reduce or eliminate symptoms of the disorder ordisease to be treated, and wherein the peptide or peptidomimeticaccording to the invention and the second pharmaceutical agent actsynergistically to reduce or eliminate symptoms of the disorder ordisease to be treated. Such second pharmaceutical agent may or may notact as a modulator of the metallopeptidase.

[0138] “Synergistically” means that the peptide or the peptidomimeticand the second pharmaceutical agent together are more effective inreducing or eliminating symptoms of a disorder or disease than eitherone alone would be at the same concentration.

[0139] The present invention also relates to a molecular complexcomprising:

[0140] the NEP receptor or the SMR1 -binding site of the NEP receptor;

[0141] the SMR1-peptide.

[0142] The present invention is illustrated in details in the followingexamples without being in any way limited in scope to these specificembodiments.

BRIEF DESCRIPTION OF THE DRAWINGS:

[0143]FIG. 1-A : Influence of spinal cord membrane protein concentrationon Substance P hydrolysis (25 nM) in the presence or absence of thesynthetic NEP inhibitor, Phosphoramidon, 10 μM. Each point representsthe percent of 3H-substance P hydrolyzed by spinal cord membraneincubated 15 min. at 30° C. in a 250 μl final volume of Tris/HCl buffer.

[0144]FIG. 1-B: Time course of Substance P hydrolysis (12.5 nM) by ratspinal cord membrane preparations in the presence or absence ofdifferent peptidase inhibitors at 10 μM final concentration:—an ACEinhibitor, captopril,—the CPB and DPPIV inhibitors, GEMSA and DPPIVinhibitor. Each point represents the percent of 3H-substance Phydrolyzed by 250 μg membrane proteins incubated at 25° C. in a 250 μlfinal volume of Tris/HCl buffer.

[0145]FIG. 2: Met-enkephalinase activity in spinal cord slices, in thepresence or absence of different peptidase inhibitors at 10 μM finalconcentration:—a NEP inhibitor, Phosphoramidon,—a NEP inhibitor,Thiorphan,—the CPB and DPPIV inhibitors, GEMSA and DPPIV inhibitor,—theSMR1 -QHNPR alone or combined with CPB and DPPIV inhibitors. Controlrepresents the Met-enkephalin recovery in the absence of tissue slice.

[0146]FIG. 2-A : Values represent the concentration of intact andimmunoreactive Met-enkephalin (mean of 2 determinations) determined byRIA analysis (μM) and recovered after 20 min. incubation at 25 ° C. with1 mg fresh tissue slices in a 1 ml final volume of KRBG buffer.

[0147]FIG. 2-B : Values represent the quantity of intact Met-enkephalin(mean of 2 determinations) determined by RP-HPLC analysis (peak heightat 18.9 min. Retention time) recovered after 20 min. incubation at 25°C. with 1 mg fresh tissue slices in a 1 ml final volume of KRBG buffer.

[0148]FIG. 3-A: Substance P hydrolysis (25 nM) by rat spinal cordslices, in the presence or absence of different peptidase inhibitors at10 μM final concentration:—a NEP inhibitor, Phosphoramidon,—a NEPinhibitor, Thiorphan,—the CPB and DPPIV inhibitors, GEMSA and DPPIVinhibitor,—the SMR1-QHNPR alone or combined with CPB and DPPIV.inhibitors. Control represents the 3H-substance P hydrolysis in absenceof tissue slice. Each point represents the percent of 3H substance Phydrolyzed by 1 mg fresh tissue slices incubated at 25° C. in a 1 mlfinal volume of KRBG buffer.

[0149]FIG. 3-B : Concentration-dependent inhibition by SMR1 QHNPR of3H-Substance P (12.5 nM) catabolism by rat spinal cord membranepreparations. Comparison with—a NEP inhibitor, Phosphoramidon and,—CPBand DPPIV inhibitors, GEMSA+DPPIV inhibitor. Comparison between theinhibitory activity exerted by QHNPR peptide alone or in combinationwith CPB and DPPIV inhibitors. Each point represents the mean recovery(in percentages) of intact 3H-substance P after 10 min; incubation at25° C. with 250 μg membrane protein in 250 μl Tris/HCl buffer (mean of 2determinations).

[0150]FIG. 4: SubstanceP-catalytic activity of various peripheral rattissue membranes and dose response inhibitory potency of SMR1-QHNPR(sialorphin) on renal membranes.

[0151]FIG. 4-A: Endopeptidase activity of the tissue membranepreparations was determined using 25 nM [3H] substanceP, in the presenceof 10 μM bestatin. The enzymatic specific activity expressed inpM/min/μg membrane protein is done in the absence and in the presence of10 μM sialorphin or NEP inhibitors (10 μM phosphoramidon or 1 μMthiorphan).

[0152]FIG. 4-B: Concentration-dependent inhibition by sialorphin of [3H]substanceP catabolism by rat renal membrane preparations. Each pointrepresents the percentage of 320 nM intact [3H] substanceP recovered andcalculated as percentage of velocity without inhibitor—velocity inpresence of inhibitor/velocity without inhibitor, and (*) represents themean±SD of four determinations. Sialorphin concentration is expressed innM and plotted on a log scale in B right. The protein concentration ofmembrane enzyme were defined according to conditions of measurement ofinitial velocity.

[0153]FIG. 5: Representative profile of untransformed (FIG. 5-A) anddouble reciprocal (FIG. 5-B) and Dixon (FIG. 5-C) plot analysis of theinhibitory activity of sialorphin on substance P-endoproteolyticactivity by rat renal membranes.

[0154] The inhibitory potency of sialophin was measured by usingsubstance P as substrate at concentrations indicated for A and B, and at14-24 nM (blank triangle) or 56-105 nM (black triangle) for FIG. 5-C.Concentrations of inhibitor for FIG. 5-A and FIG. 5-B were 0 (blackcircles), 1500 (blank circle) and 4500 nM (blank diamond). Each point ofthe untransformed and double reciprocal plots represents the mean of 2independant determinations of duplicate. Experiments were performed at25° C. in 250 μl Tris-HCl buffer (50 mM pH 7.4) under initial velocitymeasurement conditions.

[0155]FIG. 6: Pain responsiveness to a noxious stimulus in ratsfollowing intravenous administration of sialorphin.

[0156] The 3-min test session was performed 5 min after intravenous. rattail vein administration of sialorphin or its vehicle. Naloxone (0.3 mgper kg body weight) was injected subcutaneously 15 min before sialorphinadministration. Time in centre field sector (FIG. 6-C) (central squareof the open-field without pin) and activity (FIGS. 6-A and 6-B) inperipheral squares (overlayed with pins) during the 3-min test session.Each value represents the mean±sem of 8 animals per group. 1=number ofcrossings of peripheral squares; 2=rearing number on peripheral squares;3=number of escapes responses; 4=number of audible vocalisation and5=time displayed in the central area. Control group corresponded to painresponse in vehicle-treated rats. 50 μg/kg sialorphin, 100 μg/kgsialorphin, 100 μg/kg sialorphin plus naloxone 0.3 mg/kg.

EXAMPLES: Example 1 Ex vivo. Exploration of the Functional ConsequencesResulting from the Interaction of SMR1-QHNPR Peptide with NEP

[0157] The consequences of the protection of exogenous NEP-sensitivepeptides by SMR1 -Pentapeptide, in the extracellular levels ofMet-Enkephalin and Substance P have been assessed using membranepreparations and fresh slices of rat nervous tissues.

[0158] 1. Materials and methods

[0159] 1.1. Animals and tissue preparations

[0160] Sexually mature (from 7 to 9 weeks) male Wistar rats (IffaCredo), were used. Up to the day of experiment, the rats were kept underconditions of constant ambient temperature (24° C.) and of cycled light(on 8 h/off 20 h) with distribution of food and water ad libitum. On theday of the experiment, the animals were sacrificed by cardiac punctureunder pentobarbital (Sanofi, 45 mg/kg body weight, i.p.) or ketamine(Imalgene 500, Rhone Merieux, 150 mg/kg body weight, i.p.) anesthesia oralternatively by carbon dioxide asphyxia.

[0161] * Slices of fresh tissue

[0162] The organs are rapidly removed, dissected on ice, freed of nervefibers and of adipose tissues and then washed in cold oxygenatedglucose—and bicarbonate—containing Krebs Ringer (KRBG) solution, whosecomposition is the following: 120 mM NaCI-5 mM KCI-1.2 mM KH₂PO₄-27.5 mMNaHCO₃-2.6 mM CaCI₂-0.67 mM MgSO₄-5.9 mM glucose. The slices of tissuesare prepared either manually with the aid of a scalpel (1-2 mm thick),or mechanically with the aid of a “Tissue Chopper” (1 mm thick). Slicesare then dispersed into reaction tubes where they are subjected to threesuccessive washes in ice-cold oxygenated KRBG. Thereafter, they are keptat 4° C. in the same buffer supplemented with 10 μM Bestatin (a membraneaminopeptidase, (APN), inhibitor, Roche) and oxygenated under anatmosphere of 95% O2-5% CO2 until used immediately, as enzyme source.

[0163] * Membrane preparations

[0164] The organs dissected out and washed in ice-cold KRBG arehomogenized at 4° C. in 10 volumes (vol./wt.) of 50 mM Tris/HCI bufferedat pH 7.2, using a Teflon-glass homogenizer (3×5 sec.). A firstcentrifugation of 5 min. at 1000×g and 4° C. makes it possible to removethe tissular debris and the nuclei in the pellet. A secondcentrifugation of the supernatant at 100 000×g and 5° C. concentratesthe membrane fraction into the pellet, which will be superficiallywashed three times with cold Tris/HCI buffer and resuspended in freshbuffer using a Kontes homogenizer, aliquoted and stored at −80° C. whilewaiting to be used as enzyme source, at least until three months.

[0165] 1.2 Protein determination

[0166] For the determination of the tissue and membrane proteinconcentrations, the Bio-Rad DC protein assay (Bio-Rad), was used. Aswith the Lowry assay, the Bio-Rad kit is based on the reaction of sampleprotein content with an alkaline copper tattrate solution and Folinreagent. The absorbance is read at 750 nm from 15 min. to 2 h. after theaddition of reagent. The calibration curve is prepared from dilutions ofa standard solution of BSA (Bovine Serum Albumin) from 0.2 to 1.44 mg/mlprotein.

[0167] 1.3. Measurement of the NEP enzymatic activity

[0168] 1.3.1. NEP source—substrates and inhibitors

[0169] For the experiments of analysis of the NEP peptidase activity, anex vivo model using incubations of membrane and fresh tissue slicepreparations from nervous tissues that are known to be appropriate forexploring NEP peptidase activity: i.e. the dorsal zone of rat spinalcord, was first developed. The metabolism rate of the NEP-sensitivepeptides was measured using the both NEP substrates involved in thesignaling of the nociceptive response: the neuropeptides Met-enkephalinand Substance P. Native Met-enkephalin (Peninsula, 10 μM) and modifiedtritiated Substance P: [(3,4³H) Pro²-Sar⁹-Met(O₂)¹¹]-Substance P with aspecific radioactivity of 40 Ci/mmol. (NEN, 12.5-25 nM) were used.

[0170] The objective was to measure the NEP-specific endoproteolysis ofthese substrates. For that, in each test, the hydrolysis of substrateboth in the presence and in the absence of specific synthetic inhibitorsof NEP (10 μM Phosphoramidon, Roche and/or 1-10 μM Thiorphan, Sigma),and in all cases in the presence of an inhibitor of APN, the Bestatin(10 μM) was analysed. Furthermore, for studying the functional role ofSMR1-QHNPR, the reaction was carried out in the presence of theSMR1-peptide alone or combined with specific inhibitors of membranepeptidases which could inactivate the QBNPR peptide by cleaving itsC-terminal end: an inhibitor of carboxypeptidase B (GEMSA, 10 μM, Sigma)and an inhibitor of dipeptidylpeptidase IV (DPPIV inhibitor, 10 μM,Roche).

[0171] 1.3.2. The enzymatic activity assay

[0172] * Slices of fresh tissue

[0173] In the first instance, sections of fresh tissue are preincubatedin KRBG medium containing 10 μM bestatin, at 25, 30 or 37° C. in aconstantly shaken water bath and under an atmosphere of 95% O2-5% CO2,in the presence or in the absence of NEP inhibitor. At the end of thepreincubation period (15 min.), the medium is replaced with fresh mediumcontaining the substrate alone or combined with NEP inhibitor orSMR1-QHNPR and the incubation is carried out at the same incubationconditions as the preincubation. At the end of the incubation period(from 5 to 30 min.), the medium is transferred to ice-cold tubescontaining hydrochloric acid, such as the final concentration of HClwill be 0.1 N. Samples are kept at −30° C. until the measurement oftheir intact substrate and its metabolites content.

[0174] The temperature and time of incubation as well as theconcentration of substrate and of tissue enzyme are defined according tothe results such as the NEP hydrolysis activity will be measured underconditions of initial velocity.

[0175] * Membrane preparations

[0176] The membrane preparations are preincubated in 50 mM Tris/HClbuffered at pH 7.2 and containing 10 μM Bestatin, at 25, 30 or 37° C. inconstantly shaken water, in the presence or in the absence of NEPinhibitor. At the end of the preincubation period (10 min), thesubstrate is added alone or combined with NEP inhibitor orSMR1-QHNPR—and the incubation is carried out at the same incubationconditions as the preincubation. At the end of the incubation period,the reaction is stopped by cooling to 4° C. and adding to hydrochloricacid such as the final concentration of HCI will be 0.3 N. Samples arekept at −30° C. until the measurement of their intact substrate and itsmetabolites content.

[0177] The temperature and the time of the incubation as well as theconcentration of substrate and of membrane enzyme are defined accordingto the results such as the NEP hydrolysis activity will be measuredunder conditions of initial velocity.

[0178] 1.3.3. The detection of the substrate and its metabolites

[0179] To separate, detect and quantify the intact substrate and itsmetabolites, various techniques (depending on whether the substrate wasradiolabeled or not), were used: two are based on the principle ofreverse-phase chromatography for the selective isolation of the productsof the reaction (C-18 Sep-Pak cartridges and RP-HPLC) and the third isbased on the specific detection of the substrate by radio-immunoassay(RIA).

[0180] * The C-18 Sep-Pak cartridges

[0181] The C-18 Sep-Pak cartridges (Waters) were used to analyze thehydrolysis of the radiolabeled peptides: they separate compoundsaccording to their differences in polarity. This solid-phase extractionprocedure allows isolating the substrate from its metabolites, since thehydrophobic character of the peptide metabolites is reduced or even lostcompared to the intact peptide substrate.

[0182] 3H-Metabolites of radiolabeled substance P are eluted in twosteps: one with H₂0-0.1% TFA and the second one with 20% methanol −0.1%TFA, while the intact 3H-substance P is eluted in the third step with70-100% methanol -0.1% TFA. The radioactivity of eluted and isolatedcompounds is determined by liquid scintillation spectrometry.

[0183] * RP-HPLC (Reverse Phase High Performance Liquid Chromatography)

[0184] HPLC is a highly resolutive procedure that allows to isolate, anddetect by coupled spectrophotometer analysis, the non-radioactivepeptides whose concentration is at least 1 to 10 μM. The C-18 RP-HPLC isbased on the same principle as the C-18 Sep-Pak cartridge. Thechromatographic analyses were used to study the hydrolysis ofMet-Enkephalin, that were done on a C-18 LUNA analytical column (150×4.6mm inner diameter, AIT) packed with 5 μm-diameter beads.

[0185] RP-HPLC performed with a one-step 30-minute linear gradientranging from H2O-0.1% TFA to 100% acetonitril -0.1% TFA, at a 1 ml/minflow rate, leads to a resolutive separation of the two Met-Enkephalinmetabolites and of the intact substrate. Their identification andrelative quantification (peak height) are checked by continuouslymonitoring the UV absorbance at 254 nm of column outflow.

[0186] * RIA assay (Radio-Inmuno-Assay)

[0187] RIA is a fine analytical method, which allows quantifyingcompounds, whose concentration is between 1 and 100 nM or even less.Herein, a competitive RIA system has been used: the quantity ofradioactive antigen bound to the antibody decreases in a mannerinversely proportional to the quantity of antigen contained in thestandard solution or in the sample. The free radioactive antigen isseparated from the radioactive antigen—antibody complex byimmuno-precipitation.

[0188] The activity of enkephalinase NEP is monitored by quantificationof the disappearance of the initial Met-enkephalin substrate. The firstantibody used is a rabbit antibody directed against the C-terminal endof Met-enkephalin (cross-reactivity with metabolites or other peptidesis 1%) (Goros et al, J. Neurochem. (1978), 31; 29-39. Radio immunoassayof methionine and leucine enkephalins in regions of rat brain andcomparison with endorphins estimated by a radioreceptor assay). Thesecond antibody is a horse antibody directed against the rabbitimmunoglobulins. The radiolabeled antigen is iodinated Met-enkephalin(¹²⁵I-Met-Enk enkephalin) with a specific radioactivity estimated at3000 Ci/mmol.

[0189] Briefly, the Met-enkephalin RIA is performed in 100 mM Tns/HClbuffered at pH 8.6 and containing 0.1% BSA and 0.1% Triton×100. Standard(1-100 nM) or sample (100 μl), diluted anti-Met-Enkephalin antibody(100μl, {fraction (1/2000)}) and ¹²⁵I-Met-Enk (10000 cpm, 100 μl) areincubated overnight at 4° C. Bound and free fractions are separated byimmunoprecipitation with the second anti-rabbit-immunoglobulin inpresence of polyethylene glycol 6000 (6%). After centrifugation thebound radioactivity of the precipitate is quantified using agamma-spectrometer.

[0190] 2. Results

[0191] To specify the inhibitory role of the SMR1-QHNPR on the NEPenzymatic activity, it was necessary to first develop an experimentalprotocol allowing to perform the hydrolysis of Substance P orMet-Enkephalin peptides under conditions of initial velocitymeasurement.

[0192] 2.1. Search for experimental conditions of initial velocitymeasurement of NEP endopeptidase activity

[0193] 2.1.1. Hydrolysis of native Met-Enkephalin

[0194] In first series of experiment, the slices and the membranepreparations of spinal cord tissues were incubated at 30° C. in a 1 mlfinal volume of KRBG, and at 37° C. in a 0.25 ml final volume ofTris/HCl 50 mM, pH 7.2, respectively.

[0195] * RP-HPLC analysis

[0196] The calibration of the RP-HPLC chromatographic system revealsthat marker Met-enkephalin is eluted at a retention time of 18.8 min. Inthe case of the samples, a peak is identified whose height increasesconsiderably in the presence of a NEP-specific inhibitor: this peak,whose retention time is 18.8±0.2 min., corresponds to the intactMet-enkephalin substrate. Conversely, two peaks having retention timesof 5.8±0.2 min. and 12.8±0.1 min., corresponding to the metabolitesTyr-Gly-Gly and Phe-Met respectively, appear in the absence ofNEP-inhibitors. This result indicates that spinal tissue enzyme hascleaved the substrate predominantly at the Gly³-Phe⁴ amide bond of thepeptide, which already corresponds to enkephalinase activity.

[0197] At the level of membrane preparations as well as of fresh tissueslices, a high NEP-specific hydrolysis of the exogenous Met-enkephalinis observed during the 10 min. incubation at 37° C.: the spinal cordenkephalinase activity provokes a disappearance of the Met-enkephalinpeak and that is reversed in the presence of 10 μM Phosphoramidon or 1μM Thiorphan (80-90% inhibition). In addition, under these conditions,both specific NEP inhibitors ensure the almost complete inhibition ofenkephalinase activity over the time of incubation at 37° C., from 10 to30 min.

[0198] Since, the maximum hydrolysis was undoubtedly reached, at 37° C.temperature within the 10 min. incubation, in the next experiments theincubation temperature has been subsequently reduced to 30° C. then to25° C. Effectively, for the fresh tissue slices incubated at 30° C., thelevel of hydrolysis of Met-enkephalin increases with time (from 0 to 30min.). In the same manner, for the membrane preparations incubated at30° C., it is also possible to note an increase in the level ofhydrolysis in relation to the enzyme concentration (from 0 to 2 mg/ml).However, no clear linear relationship could be established.

[0199] Indeed, the HPLC chromatography coupled to spectrophotometeranalysis is a semi-quantitative technique and the single measurement ofthe heights or areas of peaks is not sufficiently precise to calculatequantitative proportional relationships. Then, to precisely quantify theMet-enkephalin, a specific quantitative RIA detection was used.

[0200] 2.1.2. Hydrolysis of modified tritiated substance P

[0201] The experimental parameters which allow to study, underconditions of initial velocity measurement, the hydrolysis of thesubstrates, Met-enkephalin and Substance P, by nervous tissue-containingNEP, have been established.

[0202] In that respect, the influence of the membrane proteinconcentration of rat spinal cord (from 0.03 to 1 mg/ml, finalconcentration) on the level of the Substance P hydrolysis (25 nM), after15 min. incubation at 30° C., was first tested. As illustrated in FIG.1-A, the levels of the 3H-Substance P degradation, expressed in percentof initial substrate concentration, increase proportionally from 2 to25% in a linear related-function to membrane protein concentration. Aclose correlation of r=0.98, n=7 was found in the absence and, ofr=0.99, n=7-7 in the presence of 10 μM Phosphoramidon. Furthermore, inthe same experimental condition, the addition of Phosphoramidon resultsin a clear reduction of the Substance P degradation (50 to 65%protection of exogenous peptide).

[0203] Similarly, the level of Substance P hydrolysis (12.5 nM) as afunction of the incubation time at 25° C. (5-20 min) was also studied.The membrane protein concentration chosen was 1 mg/ml. The Substance Pcatabolism by spinal cord membranes increases linearly with the time ofincubation, with a close correlation of r=0.97, n=18 (FIG. 1-B).Captopril, (10 μM) a potent inhibitor of the Angiotensin ConvertingEnzyme (ACE) which also cleaves the Substance P, has no effect on theactivity of the enzyme membrane preparations, as well as, for the potentinhibitors of CPB and DPPIV enzymes (protective compounds of theC-terminal SMR1 -QHNPR potential catabolism).

[0204] The conditions of initial velocity measurement of the Substance Phydrolysis by spinal cord tissue containing-NEP therefore appear to beestablished. However, the activity of both NEP inhibitors(Phosphoramidon and Thiorphan), does not appeared to be proportionallystable as a function of the incubation duration. Accordingly, the effectof the SMR1-QHNPR peptide on the NEP activity will be systematicallystudied in relation to the time of incubation.

[0205] 2.1.3. Record

[0206] The experimental conditions that allows to study, under initialvelocity measurement, the Met-enkephalin and Substance P catabolism byspinal tissues ex vivo, are reported in the table hereunder.Preincubation time  10 min (membrane preparations)  15 min (fresh tissueslices) Incubation times  5 min to 30 min. Temperature  25° C. Finalconcentration of membrane  1 mg/ml or tissue protein (spinal cord)Substrate concentration Substance P: 12.5 nM Met-enkephalin 10 μM (HPLC) 20 nM (RIA) Reaction volume  1 ml (fresh tissue slices) 250 μl(membrane preparation) Technique for separating the Sep-Pak + Liquidscintillation counter Metabolites (3H-Substance P) RP-HPLC and RIA(Met-enkephalin) Buffer Trls.HCI 50 mM, pH 7.2 + BSA  0.1% + Bestatln 10μM (membrane preparations) KRBG + BSA 0.1% + Bestatin 10 μM Oxygenatedunder 95% 02-5% CO₂ (Fresh tissue slices)

[0207] 2.2 Study of the functional consequences resulting from theinteraction of the SMR1-QHNPR peptide with NEP

[0208] 2.2.1 Degradation of Met-enkephalin by NEP spinal cord

[0209] The effect of a fixed concentration of SMR1-QHNPR (10 μM) on theMet-enkephalinase activity of spinal cord slices under experimentalconditions defined in paragraph 2.1.3, was first tested.

[0210] * RP-HPLC analysis

[0211] As illustrated in FIG. 2-B, the HPLC analyses show a strongNEP-specific hydrolysis of the Met-enkephalin substrate by spinal cordslices within the 20 min. incubation at 25° C. Phosphoramidon at aconcentration of 10 μM ensures the complete inhibition ofMet-enkephalinase activity and addition of Thiorphan (10 μM) results ina clear reduction by 80% of the Met-enkephalin degradation.

[0212] In the same experiment, the QHNPR peptide, at 10 μMconcentration, alone or combined with the inhibitors of CPB and DPPIVproteases, has an inhibitory activity of 70 or 80%; thus theSMR1-Pentapeptide is able to enter into competition with the enkephalin-pentapeptide for the NEP binding sites, both being in equalconcentrations. As in case of Substance P degradation by spinal membranepreparations, the irhibitors of CPB and DDPIV alone do not have anyintrinsic inhibitory activity on the Met-enkephalin degradation by freshspinal slices. Furthermore, they apparently are no need for protectingSMR1-QHNPR itself, especially at its C-terminal end, from the peptidaseactivity potentially present in slices of fresh spinal tissue.

[0213] In order to finely quantify the NEP activity and inhibition, thesame experiment has been analyzed with the aid of the specificMet-Enkephalin RIA.

[0214] * RIA assay

[0215] As a whole, the crude results obtained by the reverse phase-HPLCtechnique are confirmed by those derived from RIA assay (FIG. 2-A).Within the 20 min incubation period at 25° C., the Phosphoramidon,Thiorphan, as well as SMR1-QHNPR appear as very potent compounds forprotecting Met-enkephalin from NEP degrading activity. Thus, atconcentration of 10 μM, they almost totally prevented the degradation of10 μM Met-enkephalin by fresh spinal cord tissue: 96%, 100% and 96%protection, respectively.

[0216] In conclusion, all these results show the negative regulatoryrole exerted by the SMR1-QHNPR peptide on the Met-enkephalinase activityof rat nerve tissues, ex vivo.

[0217] 2.2.2 Degradation of Substance P by NEP spinal cord

[0218] * SMR1-QHNPR, an inhibitor of the NEP activity on Substance Pcatabolism

[0219] In a first instance, the effect of QHNPR peptide on thehydrolysis of Substance P was searched as it was already done inrelation to Met-enkephalin. For that, spinal cord slices were used and akinetic over a 30-min. incubation period was performed under theconditions of initial velocity measurement defined in 2.1.3.

[0220] As illustrated in FIG. 3-A, Substance P hydrolysis reactioneffectively takes place under initial velocity conditions: a closerelationships of r=0.99 was found between the percentage of Substance Phydrolysis and the incubation time at 25° C. Ten μM Phosphoramidon or 10μM Thiorphan exhibits relatively the same inhibitory activity (60-65%inhibition). The QHNPR peptide (10 μM) is found to be an efficientinhibitor: 75% inhibition of Substance P degradation when it is alone,more than 90% when it is combined with GEMBA (10 μM) and DPPIV inhibitor(10 μM). These latter, however, appear to exhibit an inherent inhibitingactivity of Substance P degradation by fresh spinal tissue.

[0221] Otherwise, in this experiment, the effect of inhibitors isproportionally stable as a function of the duration of incubation overthe 30 min. incubation period (r=0.99).

[0222] * Determination of the IC₅₀

[0223] The dose-response curve of the SMR1-QHNPR inhibitory effect on3H-Substance P degradation by spinal cord membrane preparations, shownin FIG. 3-B right panel, allows the calculation of an IC50 value(concentration of the inhibitor reducing by half the degradation of3H-substance P) of about 1.10⁻⁷ M. In the same experiment, comparisonwith Phosphoramidon reveals that protection of the exogenous Substance Pby SMR1-QHNPR is still equivalent t0 that obtained with Phosphoramidon(FIG. 3-B left panel). Furthermore, the QHNPR peptide combined with theinhibitors of CPB and DPPIV exhibits a very high NEP inhibitingactivity, greater than that of phosphoramidon (FIG. 3-B, left panel).

[0224] 2.2.3. Record

[0225] The metabolism rate of the NEP-sensitive peptides has beenmeasured using tritiated substrate coupled to chromatographic analysis(Substance P) or using native substrate coupled to specific-RIAquantification (Met-enkephalin). Under conditions of initial velocitymeasurement of the NEP enzymatic activity, an almost complete inhibitionof exogenous Met-enkephalin or Substance P catabolism resulting fromaddition of SMR1 -Pentapeptide has been observed: the concentration ofSMR1-QHNPR which reduces by half the degradation of Substance P byspinal cord tissues, was calculated to be 1.10⁻⁷M and its inhibitorypotency is equivalent to that of two well-known NEP-specific inhibitors,Thiorphan and Phosphoramidon. From these results it appears that, exvivo, the SMR1-Pentapeptide efficiently prevents the spinal NEP-induceddegradation of both neuropeptides involved in the control of spinal painperception, e.g. Substance P and Met-Enkephalin.

[0226] Example 2

SMR1-QHNPR (Sialorphn), an Inhibitor of the Substance P-Catabolism byPeripheral Tissues

[0227] The first results showed the regulatory role exerted by thesialorphin peptide on the enkephalinase activity of rat nerve tissues.The same approach was applied to peripheral tissue membrane preparationsthat are known to be enriched in NEP-peptidase and/or to be targets forsialorphin, in vivo, i.e., renal outer medulla, intestine mucosa,placenta, prostate, dental and bone tissues, as well as submandibularepithelium (Rougeot, C. et al, American Journal of Physiology, (1997)273, R1309-20; Sales et al., (1991) Regulatory Peptides 33, 209-22) andreviewed by Kenny et al., (1987), Mammalian ectoenzymes 169-210. Thereis evidence that, almost all these tissues contain substance P releasedfrom peripheral parasympathetic and sensory nerve terminals acting nearthe site of release on target cells that contain the neurokininreceptors to modulate the particular tissue function (McCarson et al.,(1999), Neuroscience 93, 361-70). Thus, it appeared that theneuropeptide could be regarded as a relevant biologically NEP substrateat the periphery. However, substance P is cleaved potently by NEP andACE membrane-bound peptidases, and both enzymes are highly distributedin the renal epithelium (Skidgel et al., (1985), Progress in Clinical &Biological Research 192, 371-8).

[0228] The specificity of the peptidase assay was assessed by testingthe inhibitory efficacy of selective peptidase inhibitors (at 1-10 μMfinal concentration to induce maximum inhibitory response) on theendoproteolysis of 3H substance P by the various tissue-membraneenzymes, and by analysing the selective formation of the NEP-relatedtritiated product of the reaction that was defined, as above usingspinal tissue. In addition, under standard conditions of initialvelocity measurement, bestatin (10 μM) was added in the incubationmedium to prevent unselectively the membrane aminopeptidase activities.

[0229] As shown in FIG. 4-A and in agreement with previous data, malerat kidney contained the highest level of substance-P-hydrolyticspecific activity: 197 pM/min/μg membrane protein from which 61±10%, n=4was due to NEP activity and 38±12% was the result of ACE activity.Sialorphin inhibited the renal membrane activity with equaleffectiveness than the phosphoramidon NEP inhibitor, i.e., 60±5% ofmaximum inhibitory response (n=9). When inhibitory efficacy was testedon purified rabbit renal NEP by using substance P as substrate (Vi=140pM/min/μg enzyme), it has been found that the substance P catabolism bythe soluble-enzyme was already inhibited by sialorphin (46% for 5 μM).

[0230] Furthermore, the inhibitory effect of sialorphin on 3Hsubstance-P degradation by rat kidney, shown in FIG. 4-B was strictlydose-dependent (r-0.970, n=20) thus allowing to evaluate the inhibitorypotency with IC50 values in the [0.5-1] micromolar range. Thisinhibitory potency is closely related to that obtained using purifiedrabbit renal NEP and synthetic specific fluorogenic substrate, i.e. 0.6μM or using rat spinal NEP, i.e. 0.4 μM.

[0231] All these results indicated that the renal NEP/sialorphinmolecular interaction that has been already evidenced after in vivotissue uptake might lead to a physiological action, for instanceprotection of the NEP-induced metabolism of regulatory peptides presentwithin this tissue, such as substance P, an humoralvasodilatory-proinflammatory mediator and autonomic neurotransmitter.Kidney that contains the highest NEP activity seems to be also a majorsite of ANP metabolism (Webb et al., (1989), Journal of CardiovascularPharmacology 14, 285-93). Thus, one can hypothesise that at renal sitessialorphin could also play a role in potentiating the physiologicaleffects of this peptide messenger whose action is clearly regulated byNEP (Kenny et al. (1988) FEBS Letters 232, 1-8), ANP is a vasodilatoryand natriuretic factor mediating physiological regulation of bloodpressure, body fluid circulation and mineral homeostasis.

[0232] In rat placenta and prostate tissues, two other peripheraltissues richest in NEP, the levels of substanceP-endoproteolyticactivity was 12-14-fold lower than in kidney and 74±10%, n=5 was shownto be due to NEP while only 8% was the result of ACE. Moreover,sialorphin decreased substance P degradation from these tissues by 70±3%. Prostate tissue was not seen to be accessible to a systemicallyadministered hydrophilic compound such as sialorphin. 3H-peptide,however this tissue is already able to synthesise it, suggesting apotential important local role for sialorphin, as inhibitor ofendogenous peptidergic signal inactivation, such as substance P (Rougeotet al., (1994), European Journal of Biochemistry 219, 765-73; Rougeot,C. et al. (1997), American Journal of Physiology 273, R1309-20).

[0233] The most striking result comes from the observation that the ratinner dental tissue which is one of major targets for sialorphin, invivo, showed high levels of substance-P endoproteolytic activity, i.e.44 pM/min/ μg membrane protein (Rougeot, C. et al. (1997), AmericanJournal of Physiology 273, R1309-20. The addition of NEP inhibitorsreduced 3H-substance P catabolic process by 53±4%, while ACE inhibitorreduced it by 21% and the sialorphin by 39±14%, n=4. In line with thisresult, is the present demonstration of sialorphin inhibitory efficacyby 75±10%, n=A on the ectopeptidase-sensitive substance P degradation byinner bone tissues. The possible involvement of sialorphin in NEPfunction within these tissues is supported by the observation that theenzyme's localisation and activity in rat peripheral tissues wellcoincide with the tissue distribution and the density of sequestrationsites for sialorphin; and these tissues included the skeletal andalveolar bones and the periosteal surfaces (Rougeot, C. et al. (1997),American Journal of Physiology 273, R 1309-20; Sales et al., (1991),Regulatory Peptides 33, 209-22; Llorens et al., (1981), European Journalof Pharmacology 69, 113-6). Furthermore, from the inner bone and dentalmembrane extracts the major sialorphin-associated molecule exhibit a pIof 5.2±0.4, (n=5) and 5.7±0.6, (n=3), respectively, thus wellcorrelating to the pI for NEP (5.5). However the physiologicalNEP-sensitive effector peptide(s) implicated in the regulation ofskeletal and dental mineralisation and/or resorption processes remain tobe identified.

[0234] A similar situation occurs for other structures previouslydemonstrated to be labelled by NEP-inhibitor or sialorphin such as therat SMG (Rougeot, C. et al. (1997), American Journal of Physiology 273,R1309-20; Sales et al., (1991), Regulatory Peptides 33, 209-22). Indeed,the SMG level of substance-P-hydrolytic specific activity was found tobe 4.2 pM/min/μg membrane protein and 55±12%, n=4 was due to NEPactivity and 20% to ACE activity, whereas the addition of sialorphinresulted in 79% inhibition. This gland is the major site of sialorphinsynthesis where it might be thus involved in the regulation of localprotein and/or fluid secretions through modulation of activity ofsubstance P, an extremely potent sialolog compound in rat (Yu et al.,(1983), Experimental Biology & Medicine 173, 467-70).

[0235] Moreover some other richly supplied area is the gut, especiallythe intestinal wall which expresses NEP, contains substance P extrinsicsensory and enteric neurons and sialorphin uptake sites (Rougeot, C. etal. (1997), American Journal of Physiology 273, R1309-20; Holzer et al.,(1997), Pharmacology & Therapeutics 73, 219-63). The substance-Pendoproteolysis by membrane fractions of the rat intestine was found tobe 93.5 pM/min/μg membrane protein. The inhibition profile showed thatin the presence of NEP inhibitors 51% of the exogenous 3H substance Pwas saved from catabolic process, whereas addition of sialorphin inducedpowerful inhibitory response with 87±17%, n=3 protection.

[0236] Taken together, these results strongly indicate that in vitro thesialorphin efficiently prevents the endopeptidase-induced degradation ofthe neuropeptide or humoral mediator, substance P, which is availablelocally in a number of tissues where NEP and sialorphin synthesis and/oruptake are also located. This suggests that the circulating sialorphincontribute in vivo to the regulation of peripheral vasodilatory andproinflammatory actions of substance P. Furthermore, as a number ofperipheral effects of circulating ANP are under NEP regulation, One canhypothesise that sialorphin also modulates its vasorelaxant, diureticand natriuretic actions, especially at renal, intestine, bone andsubmandibular sites (Kenny et al., (1988), FEBS Letters 232, 1-8; Vargaset al., (1989), Endocrinology 125, 2527-31; Gonzalez et al., (2000),Peptides 21, 875-87.

Example 3 Sialorphin Has Kinetic Behavioural Characteristics of aCompetitive Inhibitor

[0237] In order to determine inhibitor modality, all the measures ofinitial velocity of the renal enzymatic reaction were plotted versussubstrate concentration for several fixed inhibitor concentrations orversus inhibitor concentration for fixed substrate concentrations.

[0238] The pattern of lines in the untransformed (FIG. 5-A) anddouble-reciprocal plot (FIG. 5-B) as well as Dixon plot (FIG. 5-C)analyses of the inhibition by sialorphin on [3H] substance P catabolismby renal membrane are the characteristic signature of a competitiveinhibition. Competitive inhibitors function through binding at theenzyme active site, hence competing directly with the substrate for theactive free enzyme. Hence, the competition between sialorphin andsubstance P has the kinetic effect of raising the apparent Km of theenzyme for substrate by 2-5 fold.

[0239] Otherwise, tissue-uptake of the sialorphin peptide involves acomplex molecular species, including a cation mineral element, as thepeptide was only recovered in the presence of a strong divalent metalion chelating agent (Rougeot, C. et al. (1997), American Journal ofPhysiology 273, R1309-20). Furthermore chelating-FPLC analyses showedthat the sialorphin has a selective and strong zinc-chelating group,likely involving its histidine residue. The zinc ion, an essentialcomponent of the NEP catalytic site, is a common target of syntheticpotent NEP inhibitors described elsewhere. Indeed, they were designedwith a phosphate phosphoramidon) or thiol (thiorphan) or hydroxamategroups (celatorphan) as zinc-coordinating moiety, fitting the activesite of metallopeptidase (reviewed by Roques et al., (1993),Pharmacological Reviews 45, 87-146).

[0240] Taking the kinetic behaviour of sialorphin into account inaddition to the fact that the in vivo peptide interaction with itsmembrane receptor sites involved multivalent mineral ion, one canpostulate that the sialorphin shares some structural communality withthe transition state of the reaction, thus allowing to optimiseinteractions with groups in the enzyme active site, for instance as azinc coordinating ligand.

[0241] The crystal structure determination of NEP when complexed withsialorphin would allow to gain insight into the distinctive binding modeof this natural competitive inhibitor.

Example 4 Sialorphin, a New Class of Natural Analgesic

[0242] NEP plays a pivotal role in the control of biological activity ofthe neuropeptide signals involved in conveying sensory information ofdifferent modalities-from the peripheral tissues (cutaneous, muscularand visceral areas) to multiple central and peripheral nervous systemneuronal circuits. Prominent among these mediators is substance P, asensory neurotransmitter and enkephalins, the analgesic neuromodulators(Dickenson, (1995), British Journal of Anaesthesia 75, 193-200). It isdemonstrated here below that, sialorphin potently prevents theirextracellular catabolism by rat spinal tissues, in vitro.

[0243] The importance of enkephalins in modulating nociceptiveinformation has been evidenced in pre-proenkephalin gene-deficient mice,which exhibited significant decrease in nociceptive thresholds (Konig,M. et al. (1996) Nature 383, 535-8). Conversely, using inhibitors ofmembrane-bound zinc metallopeptidases, NEP and APN which are bothinvolved in the rapid inactivation of the enkephalins, resulted inpotent analgesic responses (Chen et al., (1998) Proceedings of theNational Academy of Sciences of the United States of America 95,12028-33).

[0244] To extend the insight into the in vivo possible antinociceptiveproperty of sialorphin through enkephalin-degrading enzyme inhibition,its effects were assessed in rat model of pain, i.e. the pin pain test,(Hebert et al. (1999) Physiology & Behavior 67, 99-105) in which thevarious behavioural parameters of pain responses were recorded with a3-min cutoff time.

[0245] The in vivo activity of sialorphin was tested on the pin painassay using male rats (350-400 g, Charles Rivers). The experimentaldevice consists in an open-field (45×45×40 cm) which is divided intonine equal squares (150×150 mm), eight of them are peripheral and one iscentral. The peripheral squares are overlayed with stainless steel pins(2/cm2, length 8 mm and diameter 0.6 mm). The test consisted in placingthe rat in the central square of the open-field and recording itsdifferent behaviours (cutt-off time, 3 min). Two days before the paintest, the rats were familiarised with the experimental device withoutpins for 20 miin, so as to reduce the stress linked to the spatialneophobia. All statistical analyses were carried out using the Statview5 statistical package.

[0246] As shown in the FIG. 6-A, intravenous-administeredsialorphin-treated rats emitted less vocalisation compared tovehicle-controls and displayed locomotor and exploratory activities inthe peripheral pin-areas. For instance, 100 μg per kg body weightsialorphin produced profound analgesic response, as it inducedsignificant increase in the frequency of crossings peripheral squaresduring the course of 3-min trial: 11.13±1.43, n=8 versus control2.88±0.44, n=8, p 0.001 by ANOVA and unpaired t-tests, as well as ofrearings on peripheral squares: 3.88±0.83 versus 0.75±0.41, p 0.005. Inparallel, it induced significant decrease in the number of audiblevocalisation—(0.25±0.16 versus 7.25±3.13 p 0.05) and escape—(0.13±0.13versus 6.88±2.47, p≦0.05) responses to painful stimuli.

[0247] Hence, sialorphin-treated rats displayed powerful morphine-likelevels of analgesia, i.e., 74-97% analgesia at 100 μg/kg givenintravenously, in the pin pain tests, in rat.

[0248] Furthermore, in a second test trial, the sialorphin-effect onthese behavioural parameters of noxious response, showed in FIG. 6-B,were reversed by 42-63% by prior administration of 0.3 mg per kg bodyweight naloxone (subcutaneous-injection) a μ-opioid receptor antagonist(vocalisation parameter was 20% naloxone-reversible). In addition, asshown in FIG. 6-C, sialorphin-treated rats spent significantly less timein the central area of the open-field that is not pin-overlayed thancontrols (57.75±21.30 sec versus 155.13±14.21 sec, p=0.0019), and thisbehaviour was 56% nalaxone-reversible (112.38±17.44 sec). Thisdemonstrates that μ-opiate receptor is required for completepharmalogical sialorphin-induced analgesic effect, thus supportingendogenous opioidergic mediation of sialorphin-induced analgesia.Mu-receptor dependent opioidergic pathways have an essential role inspinal and supraspinal control of nociceptive inputs and inmorphine-induced analgesia (Besse et al., (1990) Brain Research 521,15-22; Matthes et al., (1996), Nature 383, 819-23, Sora, I. et al.,(1997) Proceedings of the National Academy of Sciences of the UnitedStates of America 94, 1544-9). Thus sialorphin might produce a part ofits analgesic effects through potentiation of endogenous μopioid-dependent pathways resulting to spinal and brain antinociception.

Example 5 Study of the Activity of the QHNPR Peptide in the AversiveLight Stimulus Avoidance Test

[0249] 1—MATERIALS AND METHODS

[0250] 1. 1—Animals

[0251] Twenty four male SPF Wistar/AF rats weighing from 300 to 320 gwere used. Upon reception, the rats were weighed, marked anddistributed, in groups of 3, into F-type polycarbonate cages. Theanimals were housed in an air-conditioned animal house at a temperatureof 22-24° C. Food and drink was available to the rats ad libitum. Theywere subjected to a 12-hour light/dark cycle (light from 8 pm to 8 am).

[0252] After a period of familiarization with the laboratory conditionsof one week, the 24 rats were randomly divided into 2 groups (n=12). Therats from the various groups were all handled in the same way and underthe same conditions.

[0253] 1.2-Materials

[0254] Device for aversive light stimulus avoidance conditioning (ALSAT)

[0255] The experimental device consists of an isolated cage (50×40×37cm) which is brightly lit and comprises two levers: one lever is active,makling it possible, when it is operated, to obtain 30 seconds ofdarkness, followed by the return of the light, whereas the other leveris inactive (not positively reinforced). Pressing the active leverduring the period of darkness does not produce further periods ofdarkness. The rat is placed in the cage for 20 minutes and the number oftimes each lever is pressed is counted during the experiment.

[0256] The test battery, composed of 4 conditioning devices, is entirelyautomated and computer-controlled. Thus, no experimenter is present inthe room during the test.

[0257] 1.3—Experimental procedure

[0258] This model uses the aversion of the rat to a brightly litenvironment. During the familiarization session and the learningsession, the rat learns to control the aversive light environment of thetest device in the context of operant conditioning: the animal learns topress the active lever in order to obtain periods of darkness.

[0259] The learning test is made up over two sessions:

[0260] Session 1, familiarization with the experimental device (day 1);

[0261] Session 2, learning test (day 2).

[0262] Variables recorded

[0263] The number of times the active lever (AL) is pressed;

[0264] The number of times the inactive lever (IL) is pressed.

[0265] 1.4—Products Products Product QHNPR 0.01 N Distilled peptideacetic acid PBS water Origin BACHEM Riedel de Haèn Fluka Chaix andSwitzerland Germany Switzerland Du Marais France Preparation Dissolvedin Diluted in method acetic acid distilled water diluted in distilledwater, and buffered with D-PBS

[0266] 1.5—Administration of products

[0267] The QHNPR peptide is suspended in a proportion of 500 μg per 5 mlof 0.01N acetic acid, and then diluted with PBS in order to beadministered at the dose of 50 μg/kg, via the i.v. route, in the dorsalcaudal vein of the rat, 1 minute before the test. Product administrationprotocol Administration before the Rats per Dose Volume test Group groupTreatment (μg/kg) Route ml/kg (minutes) Vehicle 12 Acetic acid + — I.V.0.7 1 PBS Peptide 12 FG6-005 50 I.V. 0.7 1

[0268] 1.6—Statistical analyses

[0269] A two-sided probability unpaired t-test was used to compare thelever-pressing activity of the two groups of rats.

[0270] In order to evaluate the distinction between the two levers, atwo-sided probability paired t-test was used to compare the number oftimes the active lever was pressed with the number of times the inactivelever was pressed, within each of the groups.

[0271] The results are expressed as mean±standard error of the mean(SEM).

[0272] 2—RESULTS

[0273] 2.1—Total number of times the two levers were pressed during thetest sessions

[0274] During the two test sessions, the rats treated with the QHNPRpeptide proved to be significantly less active than the control rats inthe aversive light stimulus avoidance test.

[0275] Total number of times the two levers were pressed during the testsessions (mean ± SEM) Treatment Vehicle Peptide I.V. 50 μg/kg, I.V.Unpaired t-test (n = 10) (n = 10) (two-sided prob.) Session 1 25.58 ±6.15 10.08 ± 1.98 t = 2.40; p < 0.05 Session 2 21.50 ± 5.09  5.25 ± 5.09t = 3.08; p < 0.01

[0276] 2.2—Distinction between the levers

[0277] During the two test sessions, the control rats press the activelever significantly more than the inactive lever.

[0278] This is not the case with the rats treated with the QBNPRpeptide, which make no distinction between the two levers.

[0279] Distinction between the levers during the test sessions (mean ±SEM) Treatment Vehicle Peptide I.V. 50 μg/kg, I.V. (n = 10) (n = 10)Session 1 Number of times AL pressed 14.17 ± 3.52 4.83 ± 1.02 Number oftimes IL pressed 11.42 ± 2.68 5.25 ± 1.14 Paired t-test (two-sidedprob.) t = 2.30; p < 0.05 T = 0.49; N.S. AL vs IL Session 2 Number oftimes AL pressed 12.83 ± 3.22 2.67 ± 0.47 Number of times IL pressed 8.67 ± 1.97 2.58 ± 0.94 Paired t-test (two-sided prob.) t = 2.63; p <0.05 T = 0.13; N.S. AL vs IL

[0280] 3—CONCLUSION

[0281] Under these experimental conditions, during the two testsessions, the rats treated with the QHNPR peptide prove to besignificantly less active than the control rats in the aversive lightstimulus avoidance test. Furthermore, they show no learning, since theymake no distinction between the two levers. Either these rats are lesssensitive to the nociceptive light stimulus, or they are more sensitiveto the stress of the experimental light environment. Given that it hasbeen directly observed that these rats have satisfactory locomotor andexploratory activity during the tests, it is the fact that they are lesssensitive to the aversive stimulus which explains their performance. Thepeptide thus exhibits analgesic activity. The control rats showsatisfactory activity with regard to manipulating the levers and make adistinction between the active lever and the inactive lever, both duringthe first session and during the second session.

1 8 1 5 PRT Rattus rattus MISC_FEATURE (1)..(1) X is selected from anyone amino acid 1 Xaa His Asn Pro Arg 1 5 2 5 PRT Rattus rattus 2 Gln HisAsn Pro Arg 1 5 3 6 PRT Rattus norvegicus 3 Arg Gln His Asn Pro Arg 1 54 11 PRT Rattus norvegicus 4 Val Arg Gly Pro Arg Arg Gln His Asn Pro Arg1 5 10 5 5 PRT Rattus rattus 5 Gln His Asn Leu Arg 1 5 6 6 PRT Rattusrattus 6 Arg Gln His Asn Leu Arg 1 5 7 6 PRT Mus musculus 7 Gly Gln HisGly Pro Arg 1 5 8 6 PRT Mus musculus 8 Gly Gln His Asp Pro Thr 1 5

1. A therapeutic use of a SMR1-peptide or a pharmaceutically activeamount of said SMR1-peptide, for the preparation of a therapeuticcomposition for preventing or treating diseases wherein a modulation ofthe activity of a membrane metallopeptidase is sought, in a mammal,specifically in a human.
 2. The use of claim 1 wherein saidmetallopeptidase is a membrane-zinc metallopeptidase.
 3. The useaccording to claim 2, for preventing or treating diseases whereinmodulation of NEP-induced degradation of NEP-sensitive peptides issought, in a mammal, specifically in human.
 4. The use according toclaim 3, wherein said SMR1-peptide acts by inhibiting NEP.
 5. The useaccording to claims 1 to 4, wherein said SMR1-peptide acts by inhibitingenkephalin degradation.
 6. The use according to claims 1 to 5, whereinSMR1-peptide acts as an antinocipeptive agent.
 7. The use according toclaims 1 to 6, wherein said SMR1-peptide acts as an analgesic agent. 8.The use according to claims 1 to 6, wherein said SMR1-peptide acts as anagent that controls chronic inflammatory pain.
 9. The use according toclaim 8, wherein SMR1-petide acts as an agent that controls chronicinflammatory pain, such as arthritis or inflammatory bowel disease. 10.The use according to claims 1 to 6, wherein said SMR1-peptide acts as anantidiarrheal agent.
 11. The use according to claim 3, wherein saidSMR1-peptide acts by inhibiting endogenous A II formation and substanceP, BK and ANP inactivation.
 12. The use according to claims 1 to 6 or11, wherein said SMR1-peptide acts as an antihypertensive agent.
 13. Theuse according to claims 1 to 6 or 11, wherein said SMR1-peptide acts asa natriuretic agent.
 14. The use according to claims 1 to 6 or 11,wherein said SMR1-peptide acts as a diuretic agent.
 15. The useaccording to claim 1 for the prevention or treatment of atherosclerosis.16. The use according to claim 1 for the prevention or treatment of theprocesses of malignant cell proliferation and/or dissemination.
 17. Theuse according to claim 1 as an anti-infectious agent for treatinginfections such as bacterial or viral infections.
 18. The use accordingto claim 1 for controlling immuno-inflammatory responses.
 19. The useaccording to claim 1, wherein said SMR1-peptide acts as a substitute inthe treatment of drug abuse.
 20. The use according to claims 1 to 19,wherein the therapeutic composition comprises a SMR1-peptide of formula(1) X₁QHX₂X₃X₄  (1)wherein X₁ denotes a hydrogen atom or X₁ representsan amino acid chain chosen from the following: X₁═R or G, X₁═RR, orX₁═PRR, or X₁═GPRR, or X₁═RGPRR, or X₁═VRGPRR, X₂ denotes N, G or D, X₃denotes P or L and X₄ denotes R or T.
 21. The use according to claims 1to 19, wherein the SMR1-peptide is a biologically active derivative ofthe SMR1 protein.
 22. The use according to claim 20 or 21, wherein theSMR1-peptide or one of its biologically active derivatives comprises oneor more amino acids in the D-form.
 23. The use according to claim 20 or21, wherein the SMR1-peptide or one of its biologically activederivatives peptides further comprises a substituted or modified oradded group for enhancing the bioavailability or enhancing theresistance to enzymatic degradation of said peptide or biologicallyactive derivative thereof.
 24. A process for screening ligand moleculesthat specifically bind to the NEP binding site for the QHNPRpentapeptide, comprising the steps of: a) preparing a cell culture orpreparing an organ specimen or a tissue sample containing NEP bindingsites for the QHNPR pentapeptide; b) adding the candidate molecule to betested in competition with half-saturating concentration of labeledpentapeptide; c) incubating the cell culture, organ specimen or tissuesample of step a) in the presence of the labeled candidate moleculeduring a time sufficient and under conditions for the specific bindingto take place; d) quantifying the label specifically bound to the cellculture, organ specimen or tissue sample in the presence of variousconcentrations of said candidate molecule.
 25. A process for determiningthe relative affnity of ligand molecules that specifically bind, to theNEP binding site for the QHNPR pentapeptide comprising the steps a), b),c) and d) of the process of claim 24 for each candidate molecule andfurther comprising the step e) of comparing the affinity of eachcandidate molecule quantified in step d) to the one of the othercandidate molecules.
 26. A process for determining the affinity ofligand molecules that specifically bind to the NEP binding site for theQHNPR pentapeptide, comprising the steps of: a) preparing a cell cultureor preparing an organ specimen or a tissue sample containing NEP bindingsites for the QHNPR pentapeptide; b) adding the candidate molecule whichhas previously been labeled with a radioactive or a nonradioactivelabel; c) incubating the cell culture, organ specimen or tissue sampleof step a) in the presence of the labeled candidate molecule during atime sufficient under conditions for the specific binding to take place;and d) quantifying the label specifically bound to the cell culture,organ specimen or tissue sample in the presence of variousconcentrations of labeled candidate molecule.
 27. A process forscreening ligand molecules that possess an agonist biological activityon the NEP binding site of the QHNPR pentapeptide, comprising the stepsof: a) preparing a cell culture or preparing an organ specimen or atissue sample containing NEP binding sites for the QHNPR pentapeptide;b) incubating the cell culture, organ specimen or tissue sample of stepa) at concentrations allowing measurement of NEP enzymatic activityunder initial velocity conditions in the presence of the candidatemolecule, a half-saturating concentration of QHNPR and a NEP substrateduring a time sufficient for the hydrolysis activity of the NEPsubstrate to take place under initial velocity conditions; c)quantifying the activity of the NEP present in the biological materialof step a) by measuring the levels of NEP substrate hydrolysis,respectively in the presence or in the absence of the candidate ligandmolecule and in the presence or in the absence of QHNPR.
 28. A processfor screening ligand molecules that possess an antagonist biologicalactivity on the NEP binding site of the QHNPR pentapeptide, comprisingthe steps of: a) preparing a cell culture or preparing an organ specimenor a tissue sample containing NEP binding sites for the QHNPRpentapeptide; b) incubating the cell culture, organ specimen or tissuesample of step a) at concentrations allowing measurement of NEPenzymatic activity under initial velocity conditions in the presence ofa submaximal concentration of the XQHNPR peptide, specifically the QHNPRpeptide and a NEP substrate, in the presence of the candidate moleculeduring a time sufficient for the hydrolysis of the NEP substrate to takeplace under velocity conditions; c) quantifying the hydrolysis activityof the NEP present in the biological material of step a) by measuringthe levels of NEP substrate hydrolysis, respectively in the presence orin the absence of the candidate ligand molecule and in the presence orin the absence of QHNPR.
 29. A maturation product of SMR1 protein or abiologically active derivative of the SMR1 protein or of its maturationproducts which can be obtained according to the method of claims 24 to28, provided that said biologically active derivatives does not have thestructure of formula (1) as defined in claim
 20. 30. A molecular complexcomprising: the NEP receptor or the SMR1 -binding site of the NEPreceptor; a SMR1-peptide.
 31. A biologically active derivative ofSMR1-peptide characterized by its capacity either to increase or todecrease a metallopeptidase activity or to prevent the normalinteraction between the SMR1-peptide and said metallopeptidase.
 32. Thebiologically active derivative of claim 31 characterized in that saidmetallopeptidase is a membrane-zinc metallopeptidase.
 33. Thebiologically active derivative of claim 32 characterized in that saidmembrane-zinc metallopeptidase is NEP.
 34. The use according to any ofclaims 1 to 23, wherein the SMR1-peptide is associated with a secondpharmaceutical agent that acts synergistically with the SMR1-peptide.35. Use of a biologically active derivative of the SMR1 protein formodulating the interaction between endogenous SMR1 protein or peptideand a membrane metallopeptidase.
 36. Use of an agent that modulates theinteraction between endogenous SMR1 protein or peptide and a membranemetallopeptidase for the preparation of a therapeutic composition forpreventing or treating diseases wherein a modulation of the activity ofsaid membrane metallopeptidase is sought.